Synthetic HPV11 virus-like particles

ABSTRACT

The present invention is a series of synthetic virus-like particles useful in the characterization of human papillomavirus infection and assays employing the synthetic virus-like particles. The synthetic virus-like particles are generated from constructs designated as HPV6:4; HPV6:5; HPV6:2; HPV6:4Δ132; and HPV6:4,S131G.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not applicable.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

This application claims priority from U.S. Provisional application Ser.No. 60/006,788, filed Nov. 15, 1995.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

The present invention is a series of synthetic virus-like particles(VLP) useful in the characterization of human papillomavirus infectionand assays employing the synthetic virus-like particles.

BACKGROUND OF THE INVENTION

Papillomavirus infections occur in a variety of animals, includinghumans, sheep, dogs, cats, rabbits, monkeys, snakes and cows.Papillomaviruses infect epithelial cells, generally inducing benignepithelial or fibroepithelial tumors at the site of infection.Papillomaviruses are species specific infective agents; a humanpapillomavirus cannot infect a nonhuman animal.

Papillomaviruses may be classified into distinct groups based on thehost that they infect. Human papillomaviruses (HPV) are furtherclassified into more than 60 types based on DNA sequence homology (for areview, see Papillomaviruses and Human Cancer, H. Pfister (ed.), CRCPress, Inc., 1990). Papillomavirus types appear to be type-specificimmunogens in that a neutralizing immunity to infection to one type ofpapillomavirus does not confer immunity against another type ofpapillomavirus.

In humans, different HPV types cause distinct diseases. HPV types 1, 2,3, 4, 7, 10 and 26-29 cause benign warts in both normal andimmunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25,36 and 46-50 cause flat lesions in immunocompromised individuals. HPVtypes 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata ofthe genital or respiratory mucosa. HPV types 16 and 18 cause epithelialdysplasia of the genital mucosa and are associated with the majority ofin situ and invasive carcinomas of the cervix, vagina, vulva and analcanal. HPV6 and HPV11 are the causative agents for more than 90% of allcondyloma (genital warts) and laryngeal papillomas. The most abundantsubtype of HPV type 6 is HPV6a.

Immunological studies in animals have shown that the production ofneutralizing antibodies to papillomavirus antigens prevents infectionwith the homologous virus. The development of effective papillomavirusvaccines has been slowed by difficulties associated with the cultivationof papillomaviruses in vitro. The development of an effective HPVvaccine has been particularly slowed by the absence of a suitable animalmodel. Neutralization of papillomavirus by antibodies appears to betype-specific and dependent upon conformational epitopes on the surfaceof the virus.

Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNAviruses that encode for up to eight early and two late genes. The openreading frames (ORFs) of the virus genomes are designated E1 to E7 andL1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 codefor virus capsid proteins. The early (E) genes are associated withfunctions such as viral replication and cellular transformation.

The L1 protein is the major capsid protein and has a molecular weight of55-60 kDa. L2 protein is a minor capsid protein which has a predictedmolecular weight of 55-60 kDa and an apparent molecular weight of 75-100kDa as determined by polyacrylamide gel electrophoresis. Immunologicdata suggest that most of the L2 protein is internal to the L1 protein.The L2 proteins are highly conserved among different papillomaviruses,especially the 10 basic amino acids at the C-terminus. The L1 ORF ishighly conserved among different papillomaviruses.

The L1 and L2 genes have been used to generate vaccines for theprevention and treatment of papillomavirus infections in animals. Zhouet al., (1991; 1992) cloned HPV type 16 L1 and L2 genes into a vacciniavirus vector and infected CV-1 mammalian cells with the recombinantvector to produce virus-like particles (VLP).

Bacterially-derived recombinant bovine papillomavirus L1 and L2 havebeen generated. Neutralizing sera to the recombinant bacterial proteinscross-reacted with native virus at low levels, presumably due todifferences in the conformations of the native and bacterially-derivedproteins.

Recombinant baculoviruses expressing HPV6 L1, HPV11 L1, HPV16 L1, HPV18L1, HPV31 L1 or HPV16 L2 ORFs have been used to infect insect Sf9 cellsand produce L1 and L2 proteins. Western blot analyses showed that thebaculovirus-derived L1 and L2 proteins reacted with antibody to HPV16.The baculovirus derived L1 forms VLPs.

Carter et al., (1991) demonstrated the production of HPV16 L1 and HPV16L2 proteins by recombinant strains of Saccharomyces cerevisiae. Carteret al. also demonstrated the production of HPV6b L1 and L2 proteins. TheHPV6b L1 protein was not full-length L1 protein. The recombinantproteins were produced as intracellular as well as secreted products.The recombinant L1 and L2 proteins were of molecular weights similar tothe native proteins. When the proteins were expressed intracellularly,the majority of the protein was found to be insoluble when the cellswere lysed in the absence of denaturing reagents. Although thisinsolubility may facilitate purification of the protein, it may hamperanalysis of the native epitopes of the protein.

Recombinant proteins secreted from yeast were shown to containyeast-derived carbohydrates. The presence of these N-linkedoligosaccharides may mask native epitopes. In addition, the secretedrecombinant proteins may contain other modifications, such as retentionof the secretory leader sequence.

The present invention is directed to the production of recombinantpapillomavirus proteins having the immunity-conferring properties of thenative papillomavirus proteins as well as methods for their productionand use. The present invention is a series of synthetic virus-likeparticles useful in the characterization of human papillomavirusinfection and assays employing the synthetic virus-like particles.

The invention involves the delineation of residues specific to HPV11 L1which are required for binding neutralizing antibodies. The inventionfurther involves the delineation of two residues specific to HPV11 L1which together are necessary and sufficient for binding neutralizingantibodies.

HPV11 L1 contains only 38 amino acid differences from HPV6 L1, plus oneresidue insertion. In spite of the strong identity between these twoproteins, a panel of neutralizing monoclonal antibodies which arespecific for HPV11 VLPs has been generated. We determined which of theseamino acid positions are important for the binding of the neutralizingmonoclonal antibodies. This was accomplished by assessing binding of themonoclonal antibodies to a family of HPV11 clones which containedsubstitutions of HPV6b amino acid residues for HPV11 amino acid residuesat these positions, and then mutating HPV6b to match the HPV11 sequenceat these critical positions. We demonstrated that the neutralizingantibodies will bind HPV6b VLPs with as few as two of thesesubstitutions, and that both substitutions are essential for binding.This work defines the minimal unit for binding neutralizing antibodies.

The panel of neutralizing monoclonal antibodies for HPV11 was obtainedfrom Neil Christiansen (Pennsylvania State University, Hershey, Pa.).The monoclonal antibodies in the panel are HPV11 specific andVLP-dependent. The antibodies may be distinguished from each other interms of which amino acid residues affect binding of the individualantibodies, although there are overlapping positions for all themonoclonal antibodies.

These residues collectively define the epitope for antibodies known toneutralize HPV11. In principle, the mutation of HPV6 L1 in only theseselect positions results in binding to these HPV11 specific neutralizingmonoclonal antibodies. The derivatized HPV6 VLPs may be used to generatemonoclonal antibodies to the HPV11 neutralizing epitope. This is thebasis of a release assay to verify that manufactured HPV11 VLPs containthe neutralizing epitope.

This problem has not been solved in the past and, to our knowledge, isthe first demonstration of the transfer of a conformationlly dependentepitope.

There were two difficulties to overcome. First, the epitope isconformational, and conventional means of epitope mapping, binding topeptide fragments, could not be utilized. It was necessary to expressany test L1 protein in a way that facilitated formation of virus-likeparticles which mimic the virus structure. Second, the large number ofL1 clones required for the mapping necesitated the generation of afacile means to express the test viral coat proteins.

Without knowledge of the neutralizing epitope, it would be difficult tovalidate manufacture of VLPs for commercial use.

One use of the derivatized VLP is as a reagent in a release assay toHPV11. HPV6 L1 is mutated to match HPV11 in the positions defined bythese studies. Binding of the HPV11 neutralizing monoclonal antibodiesto these derivatized HPV6 VLPs will be demonstrated.

These derivatized HPV6 VLPs may be used in a competition binding assaywith manufactured HPV11 VLPs for binding to the HPV11 neutralizingmonoclonal antibodies. Only those HPV6 derivatives demonstrated to bindthe monoclonal antibodies will compete with authentic material.

Alternatively, monoclonal antibodies may be generated to theneutralizing epitope on derivatized HPV6; then manufactured HPV11vaccine will be demonstrated to bind these antibodies.

SUMMARY OF THE INVENTION

The present invention is a series of synthetic virus-like particlesuseful in the characterization of human papillomavirus infection andassays employing the synthetic particles. The synthetic virus-likeparticles are generated from constructs designated as HPV6:4; HPV6:5;HPV11:G131S; HPV11 :Δ132; HPV11Y246F; HPV11:N278G; HPV11:S346T; HPV6:2;HPV6:4Δ132; and HPV6:4,S131G.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows VLPs with type-specific properties are generated intransient transfection. Sf9 cells were cotransfected with Baculogold™DNA and either pVL1393:CRPV or pVL1393:HPV11. Cells were harvested aftersix days, extracts prepared, and ELISAs performed as described in thetext. Column 1, CRPV VLPs; column 2, HPV11 VLPs; column 3, SF9 extract;column 4, baculovirus DNA; column 5, pVL1393:CRPV; column 6,pVL1393:HPV11.

The primary antibody is 10⁻⁵ dilution of CRPV.5A ascites fluid.

FIG. 2 shows the same experiment as FIG. 1, except that the primaryantibody is 10⁻⁵ dilution of H11.F1 ascites fluid.

FIG. 3 shows the immunogenic material produced by transient transfectionis sensitive to denaturation. Sf9 cells were cotransfected withpVL1393:HPV11 and BaculoGold™ DNA, cells were harvested after six days,and extracts prepared as described in the text. A portion of theextracts were denatured by dilution into 0.1M Sodium Carbonate, pH 10.5,and incubated at room temperature for 1 hour. These extracts were thencoated onto a microtiter plate and allowed to dry. Untreated extractswere coated onto microtiter plates and incubated overnight at 4° C.ELISAs were performed as described in Methods using a 10⁻⁵ dilution ofeither H11.F1 or H6.C6 ascites. Column 1, Sf9 extract; column 2, pVL1393extract. A, extract is non-denatured. B, extract was carbonate buffertreated.

FIG. 4 shows the amino acid sequences of the HPV11 and HPV6 L1 protein.These sequences are also available in the EMBL Gene Bank.

FIG. 5 shows that VLPs produced from clone HPV6:2 (with a substitutionat position 131, followed by the tyrosine insertion to create position132) binds antibodies H11.B2, H11.F1 and H11.G5. In contrast, VLPsproduced from HPV6:4 back-mutated at either position 131 (HPV6:4, G131S)or 132 (HPV6:4Δ132) do not bind these antibodies, in spite of thepresence of the three other critical residue changes. Antibodies H11B2,H11F1 and H11G5 are neutralizing MAbs in the xenograft system. H6C6measures the total level of L1 production as described in Example 4. TheELISA was performed as described in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a series of synthetic virus-like particles(VLP) useful in the characterization of human papillomavirus infectionand assays employing the synthetic virus-like particles, which may beused to monitor and validate VLPs manufactured through recombinant DNAtechnologies. The synthetic virus-like particles are generated fromconstructs designated as HPV6:4; HPV6:5; HPV11:G131S; HPV11:Δ132;HPV11Y246F; HPV11:N278G; HPV11:S346T; HPV6:2; HPV6:4Δ132; andHPV6:4,S131G.

Papillomavirus infections occur in a variety of animals, includinghumans, sheep, dogs, cats, rabbits, monkeys, snakes and cows.Papillomaviruses infect epithelial cells, generally inducing benignepithelial or fibroepithelial tumors at the site of infection.

Papillomaviruses may be classified into distinct groups based on thehost that they infect. Human papillomaviruses (HPV) are furtherclassified into more than 60 types based on DNA sequence homology (for areview, see Papillomaviruses and Human Cancer, H. Pfister (ed.), CRCPress, Inc., 1990). Papillomavirus types appear to be type-specificimmunogens in that a neutralizing immunity to infection to one type ofpapillomavirus does not confer immunity against another type ofpapillomavirus.

In humans, different HPV types cause distinct diseases. HPV types 1, 2,3, 4, 7, 10 and 26-29 cause benign warts in both normal andimmunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25,36 and 46-50 cause flat lesions in immunocompromised individuals. HPVtypes 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata ofthe genital and respiratory mucosa. HPV types 16 and 18 cause epithelialdysplasia of the genital tract and are associated with the majority ofin situ and invasive carcinomas of the cervix, vagina, vulva and analcanal. HPV6 and HPV11 cause the majority of genital warts and laryngealpapillomas.

Immunological studies in animals have shown that the production ofneutralizing antibodies to papillomavirus capsid proteins preventsinfection with the homologous virus. The development of effectivepapillomavirus vaccines has been slowed by difficulties associated withthe cultivation of papillomaviruses in vitro. The development of aneffective HPV vaccine has been particularly slowed by the absence of asuitable animal model. Neutralization of papillomavirus by antibodiesappears to be type-specific and dependent upon conformational epitopeson the surface of the virus.

Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNAviruses that encode for up to eight early and two late genes. The openreading frames (ORFs) of the virus genomes are designated E1 to E7 andL1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 codefor virus capsid proteins. The early (E) genes are associated withfunctions such as viral replication and transformation.

The L1 protein is the major capsid protein and has a molecular weight of55-60 kDa. L2 protein is a minor capsid protein which has a predictedmolecular weight of 55-60 kDa and an apparent molecular weight of 75-100kDa as determined by polyacrylamide gel electrophoresis.

The production of HPV 16 L1, HPV16 L2, and HPV type 6 L1 proteins byrecombinant strains of Saccharomyces cerevisiae has been reported. Itwould be useful to develop methods of producing large quantities ofpapillomavirus proteins of any species and type by cultivation ofrecombinant yeasts. It would also be useful to produce large quantitiesof papillomavirus proteins having the immunity-conferring properties ofthe native proteins, such as the conformation of the native protein. Toachieve this latter goal it would be necessary to analyze the effect ofnumerous mutations in th L1 gene on the binding of antibodies of knownproperties (VLP dependent, cross-reactive, etc.)

The empirical scanning of natural or engineered peptide sequences forfunctional residues is inherently dependent upon expression of largenumbers of sequence variants to assay their relative functional potency.The level of protein expression obtained can be particularly critical inthe case of self-assembling viral structural proteins, because theefficiency of self-assembly frequently is concentration dependent. Theinsect baculovirus expression vector system has been widely used tostudy viral self-assembly, but it generally requires prior isolation andexpansion of a plaque-purified recombinant viral stock to generateuseful quantities of self-assembled particles. In examining a number ofpossibilities for expression of analytical levels of the L1 coat proteinof Cottontail Rabbit and Human Type 11 Papillomaviruses, we found thateven brief transient cotransfection of insect cells with baculovirustransfer vectors and viral DNA yielded assembled particles which wereimmunologically indistinguishable from particles previously obtainedfrom plaque purified stocks. Within six days of plasmid/viral DNAcotransfection of Sf9 cells, at least 1-2 μg of assembled L1particles/100 mm plate could be demonstrated. This level of expressionis more than sufficient to assay functionality, and has severaladvantages over comparable mammalian cell transient expression systems.

To define neutralizing epitopes in HPV infections, we need to identifythe amino acid residues that confer antigenic type-specificity on humanpapillomavirus subtypes (Christensen, N. D., et al., 1990,. Monoclonalantibody-mediated neutralization of infectious human papillomavirus type11. J. Virol. 64, 1936-1944). Many of the type-specific epitopes areconformationally-dependent and are detectable only upon VLP assembly.The L1 structural coat protein of several animal and humanpapillomaviruses has been demonstrated to efficiently self-assemble whenexpressed in insect cells via recombinant baculovirus strains(Christensen, N. D., et al., 1994, Assembled baculovirus-expressed humanpapillomavirus type 11 L1 capsid protein virus-like particles arerecognized by neutralizing monoclonal antibodies and induce high titresof neutralizing antibodies. J. Gen. Virol. 75, 2271-2276). The time andlabor involved in the generation of recombinant phage precludes the useof this method to screen a large number of VLP variants produced throughsite-directed mutagenesis. However, we previously observed that whenexpressed in the baculovirus system, a recombinant protein is detectableas a secreted product in μg/ml quantities within 5-7 days of the initialtransfection of insect cells with plasmid and viral DNAs. Based uponthis observation, we examined whether sufficient quantities ofpapillomavirus L1 protein would accumulate to allow self-assembly intoVLPs upon transient expression, particularly if a more efficientbaculovirus transfection system such as the Baculogold™ (Pharmingen, SanDiego, Calif.) system were utilized. Employing a rapid 6-day transienttransfection protocol, the L1 coat protein of of numerous papillomavirustypes, properly assembled into VLPs, was produced. Extracts preparedfrom transiently transfected cells with CRPV or HPV11 L1 gene constructscontained immunogenic material recognized by type-specific and VLPdependent monoclonal antibodies generated against either CRPV or HPV11VLPs. The transiently expressed material was not cross-reactive withother type-specific antibodies, and recognition was sensitive toalkaline denaturation, further demonstrating fidelity in VLP formation.

To map the HPV11 neutralizing epitope, we individually mutated HPV11 atthe residues where the sequence diverges from the HPV6b sequence. Thepositions were mutated to match the HPV6b sequence (the tyrosine atposition 132 was deleted to analyze the effect of this insertion). Usingthe Sf9 transient expression system described above, these mutant HPV11L1 genes were expressed and analyzed for binding by HPV11 specificmonoclonal antibodies.

The following examples are provided to further define the inventionwithout, however, limiting the invention to the particulars of theseexamples.

EXAMPLE 1

Generation of test expression constructs.

The HPV11 L1 structural gene was cloned from clinical isolates using PCRwith primers designed from the published L1 sequence. The L1 gene wassubsequently subcloned both into BlueScript (Pharmacia) for mutagenesis,and pVL1393 (Stratagene) for expression in Sf9 cells.

Mutations were introduced into the L1 gene using Amersham Scultor invitro mutagenesis kit. The appearance of the desired mutation wasconfirmed by sequencing, and the mutated gene subcloned into pVL1393 forexpression in Sf9 cells.

The HPV6 L1 structural gene was subcloned both into pAlt-1 (Promega) formutagenesis, and pVL1393 (Stratagene) for expression in Sf9 cells.Mutations were generated using the Altered Sites II in vitro mutagenesisSystems (Promega), verified by sequencing, and subcloned into pVL1393for expression in Sf9 cells.

Sequences of the L1 genes of HPV6 and HPV11 were verified with thepublished sequences. (Dartmann, K., et al. 1993, EMBO J. 2: 2341; EMBLGeneBank Accession #M14119 (HPV11 L1) and Accession #X00203 (HPV6B L1)!.

EXAMPLE 2

Transient Expression of L1 VLPs in SF9 cells.

SF9 cells were transfected using BaculoGold Transfection kit(Pharmingen). Transfections were done essentially according to themanufacturer's instructions with the following modifications. 8·10⁸ Sf9cells were transfected in a 100 mM dish, with 4 μg of BaculoGold DNA and6 ug of test DNA. Cells were harvested after 6 days and assayed for VLPproduction.

EXAMPLE 3

Preparation of SF9 extracts and ELISA assays.

Cells were harvested six days after transfection, by scraping followedby low speed centrifugation. Cells were resuspended in 300 μl ofbreaking buffer (1M NaCl, 0.2M Tris pH 7.6) and homogenized for 30" onice using a Polytron PT 1200 B with a PT-DA 1205/2-A probe (Brinkman) ina Falcon 1259 tube. Samples were spun at 2500 rpm for 3 minutes topellet debris. Tubes were washed with an additional 150 μl of breakingbuffer, supernatents collected in a 1.5 ml microfuge tube, and respunfor 5 minutes in an Eppendorf microfuge (Brinkman). Supernatants werecollected and stored at 4° C. until use. ELISA assays typically wereperformed the same day.

5 μl of extract was diluted into 50 μl of 1% BSA in PBS (phosphatebuffered saline; 20 mM NaPO₄, pH 7.0, 150 mM NaCl) and plated onto apolystyrene plate. The plate was incubated overnight at 4° C. Extractswere removed and the plate blocked with 5% powdered milk in PBS. Allsubsequent wash steps were performed with 1% BSA in PBS. The plate wasincubated at room temperature with primary antibody for 1 hour. Primaryantibodies, monoclonal antibodies generated against HPV11 VLPs, wereobtained as ascites stock from Dr. Neil Christensen (Pennsylvania StateUniversity). They were diluted 10⁵ in 1% BSA PBS before use. Afterwashing, plates were incubated for 1 hour with secondary antibody. Thesecondary antibody, peroxidase labeled Goat anti-Mouse IgG (γ), waspurchased from Kirkegaard & Perry Laboratories, Inc. and used at 10³dilution in 1% BSA in PBS. After a final washing, a horseradishperoxidase assay was performed and absorbance read at 405 nm.

EXAMPLE 4

HPV11 scan

To map the residues critical for an HPV11 specific neutralizing epitope,we take advantage of two conditions. First of all, we used a panel ofmonoclonal antibodies which are specific for HPV11 L1 and recognize L1only when in a VLP. The assay conditions described in Example 3 are suchthat these antibodies are non-cross-reactive to the closely relatedHPV6b L1 VLP. Among these five antibodies, 4 have been demonstrated toneutralize HPV 11 in the Kreider Xenograft system (Kreider et al., 1987,J. Virol. 61: 590-593)

HPV6 and HPV11 L1s are the most closely related L1 proteins within thepapilloma virus family. HPV6 L1 is 500 amino acid residues in length.HPV11 L1 is 501 residues. They can be aligned such that the extra aminoacid in HPV11 is at position 132. With this alignment, they areidentical in amino acid sequence in all but 39 positions (92.4%),including the insertion.

We reasoned that the type 11 specificity of the monoclonal antibodiesmust reside within these 39 residue differences. By systematicallychanging a type 11 residue into a type 6, those residues critical to thetype 11 response would be revealed by a loss in binding affinity by thetype 11 specific monoclonal antibodies. Because the residues would bemutated to residues which appear naturally in type 6, the likelihood ofsuch substitutions affecting VLP formation would be small.

To determine the affect on binding of any particular residue, both HPV11and the corresponding HPV11 derivative were expressed in the transientexpression system. An ELISA was performed using the panel of HPV11specific monoclonal antibodies, and results between the two compared. L1production was normalized with monoclonal antibody H6.C6. H6.C6 antibodyis cross-reactive with HPV11, the epitope is linear and independent ofVLP formation. Thus it measures L1 production.

Results are put through a double normalization. First, the ratio ofabsorbance of the test antibody to H6.C6 is calculated for the testposition. The same ratio is determined for HPV11 and divided into theratio for the test position. Thus a double ratio near 1 means that thereis no detectable difference in antibody binding to the test clonerelative to HPV11. A double ratio less than one means that the testantibody binds more poorly to the test clone than wild-type. In theory,a ratio greater than 1 means that the antibody binds better to the testclone than to HPV11. In practice this was not observed. A ratio in therange of 0.1 to 0.2 is essentially background, meaning we cannot detectbinding of the antibody to the mutant VLP.

The positions in HPV11 L1 which differ from HPV6 were individuallymutated the match the corresponding residue in HPV6. Clones wereexpressed in SF9 cells through a Baculovirus expressing recombinant, andaffect of binding by the panel of HPV11 specific mononclonal antibodiesdetermined (Table 1). Residues which appear in column 2, labelled `lessbinding`, are positions deemed critical for binding one or more of themonoclonal antibodies. Residues listed in column 1, labeled `retainsbinding`, are judged not critical for binding any of the monoclonalantibodies.

                  TABLE 1                                                         ______________________________________                                        Retains Binding      Loses Binding                                            ______________________________________                                        HPV11:K28T           HPV11:G131S                                              HPV11:Y49F           HPV11:Y132Δ                                        HPV11:K53R           HPV11:Y246F                                              HPV11:V54A           HPV11:N278G                                              HPV11:L119F          HPV11:S346T                                              HPV11:T170K                                                                   HPV11:S173T                                                                   HPV11:S166P                                                                   HPV11:N179A                                                                   HPV11:L219I                                                                   HPV11:V225T                                                                   HPV11:T263E                                                                   HPV11:D271T                                                                   HPV11:L273I                                                                   HPV11:V274I                                                                   HPV11:G277S                                                                   HPV11:S281T                                                                   HPV11:A294G                                                                   HPV11:H300N                                                                   HPV11:H325Q                                                                   HPV11:K347T                                                                   HPV11:A349S                                                                   HPV11:F366V                                                                   HPV11:Q434P                                                                   HPV11:D439N                                                                   HPV11:M440L                                                                   HPV11:F458Y                                                                   HPV11:T474S                                                                   HPV11:A467I                                                                   HPV11:P488A                                                                   HPV11:T497A                                                                   ______________________________________                                    

The clones which encode the L1 mutants described in Table 1 under thecolumn `loses binding` are designated HPV11:G13S, HPV11:Y132Δ,HPV11:Y246F, HPV11:N278G, and HPV11:S346T. Alternatively, the `HPV`prefix may be omitted.

The four residues which affect binding are spaced at considerabledistances from each other, with the total span encompassing greater than200 residues along the linear sequence.

The positions affect the binding of the antibodies differentially. Nomore than three positions affect the binding of any single antibody.Only position 246 affects the binding of all five antibodies. In allcases, some measure of detectable binding is observed. This indicatesthat VLP formation is not affected. The affect on binding by the changeat position 278 appears marginal and is questionable, but it is includedat this time because the slight diminishment is reproducible. The affecton binding, as measured by the VLP normalized affinity ratio, is givenin Table 2. Table 3 gives the binding configurations for the HPV11monoclonal antibodies, as deduced from these studies.

                  TABLE 2                                                         ______________________________________                                        VLP Normalized Affinity Ratio*                                                Position                                                                            H11.A3.2   H11.B2  H11.F1  H11.G5                                                                              H11.F3                                 ______________________________________                                        G131S 0.93       0.20    0.11    0.12  0.96                                   Δ132                                                                          1.0        0.36    0.08    0.11  0.64                                   Y246F 0.48       0.33    0.52    0.45  0.32                                   N278G 1.4        0.79    0.69    0.81  0.92                                   S173T 0.82       1.14    0.85    0.85  0.94                                   S346T 0.98       1,6     0.74    0.79  0.32                                   ______________________________________                                         *(Normalized affinity ratio = PositionX(A.sub.405 H11.Y/A.sub.405             H6.C6)/HPV11(A.sub.405 H11.Y/A.sub.405 H6.C6)                            

                  TABLE 3                                                         ______________________________________                                        Antibody Binding Configurations                                               Position                                                                            H11.A3.2   H11.B2  H11.F1  H11.G5                                                                              H11.H3                                 ______________________________________                                        G131S +          -       -       -     +                                      Y132Δ                                                                         +          -/+     -       -/+   +                                      Y246F -/+        -/+     -/+     -/+   -/+                                    N278G +          +/-     +/-     +/-   +/-                                    S346T +          +       +       +     -                                      ______________________________________                                    

EXAMPLE 5

Stripping Assay

To monitor the production of HPV11 VLPs to insure that they contain theimportant neutralizing epitope, the following competition ELISA isemployed. HPV6 derivative VLPs, but not HPV6 VLPs, compete for bindingto HPV11 VLPs with monoclonal antibodies H11.B2, H11.F1, and H11.G5.This shows the presence of the neutralizing epitope on the VLPs bydemonstrating specific, competable binding to the neutralizing epitope.The assay is performed in the following way.

1. Plate 10-100 ng of test batch HPV11 VLPs per well of a 96 well ELISAplate. Dilute sample in 1.0% BSA in PBS (ELISA buffer). Plate 50 μlsample. Incubate overnight at 4° C.

2. Remove supernatants from wells. Block for one hour with 5% powderedmilk in PBS at room temperature.

3. Rinse with ELISA buffer.

4. Prepare dilutions of monoclonal antibody H11.F1

A. Prepare a set of dilutions with increasing amounts of HPV6 derivativeVLPs.

B. Prepare a duplicate set of dilutions with increasing amounts of HPV6VLPs.

C. Prepare a dilution with no VLPs added.

5. Add 50 μl of the antibody samples to the wells of the ELISA plate.Incubate for one hour at room temperature.

6. Remove antibodies and wash three times with ELISA buffer.

7. Add 50 μl of goat anti-mouse IgG (γ) at appropriate dilution.Incubate for one hour at room temperature.

8. Wash three times with ELISA buffer. Develop with an alkalinephosphatase assay and read at 405 nm.

9. A strong signal at 405 nm that is strongly competed with HPV6derivative VLPs, but not HPV6 VLPs will verify the pressence of theneutralizing epitope on the test batch of HPV11 VLPs.

EXAMPLE 6

Monitoring Neutralization

HPV6 derivative VLPs are used to characterize test batches of polyclonalsera for neutralizing activity. A batch of polyclonal sera is generated,for example, by a test batch of HPV11 VLPs. Alternatively, it is a humansample for which a characterization of its neutralizing capability isdesired.

A polyclonal sera is pre-cleared with HPV6 VLPs. This removescross-reactive antibodies, both VLP dependent and non-dependent. TheHPV11 neutralizing epitope is type 11 specific, and antibodies generatedagainst it are not removed by pre-incubation with HPV6 VLPs. However,derivatized HPV6 particles bind these antibodies, and observation ofsuch binding, in for example a standard ELISA, demonstrates the presenceof neutralizing antibodies in the test sera sample.

A test sample of polyclonal sera is cleared according to the followingprocedure.

1. An estimate of the total VLP binding antibody is made. VLPs will beimmobilized on an ELISA plate in sandwich format using an anti-HPV11monoclonal (several are available). The amount of polyclonal antibodywhich binds is estimated using a second anti-HPV11 antibody of knownconcentration as a standard. Alternatively, the concentration of IgG ofthe polyclonal is determined and assumed to be all anti-HPV11.

2. HPV6 VLPs are added to an aliquot of sera in 10-fold μg excess to theamount of HPV11 antibody in the polyclonal sera, as determined in stepone.

3. The mixture is incubated overnight at room temperature, followed byhigh speed centrifugation (300,000 g) for 5 hours to pellet theVLP-antibody complexes.

4. The procedure is repeated two more times.

5. The stripped sera is tested for binding in a sandwich ELISA. HPV6 andHPV6 derivative VLPs (which bind the neutralizing monoclonals) will beimmobilised by an HPV6 monoclonal antibody. The stripped polyclonal serashould show only minimal binding to HPV6 VLPs. A strong signal againstHPV6 derivitised VLPs demonstrates binding to the principal neutralizingdomain of HPV11, and that the polyclonal sera contains neutralizingantibody.

A second assay may be established to demonstrate neutralizing capabilityin test sera sample using the Xenograph neutralization assay(Christensen et al., 1990. J. Virol. 64: 1936-1944; Christensen et al.,1994, J. Gen. Virol. 75: 2271-2276).

1. Stripped sera against HPV6 derivative VLPs are generated according tothe protocol given above, substituting HPV6 derivative VLPs for HPV6VLPs. Polyclonal sera stripped with HPV6 VLPs are made as a control.

2. A series of dilutions of the polyclonal sera are made and analyzed inthe Xenograph neutralization assay to establish the neutralizing titerof the sera.

3. Parallel sets of dilutions of HPV6 derivative stripped and HPV6stripped sera are made and titered in the Xenograph.

4. The presence of neutralizing activity in the Xenograph assay that islargely removed by stripping with HPV6 derivative VLPs, but not HPV6VLPs, demonstrates by a biological assay the presence of antibodies inthe sera against the HPV11 neutralising epitope.

EXAMPLE 7

Transient expression of VLPs in Sf9 cells

The HPV11 L1 structural gene was cloned from clinical isolates using thePolymerase Chain Reaction (PCR) with primers designed from the publishedL1 sequence (8,17). The CRPV L1 structural gene was cloned by PCR fromviral genomic DNA. The L1 genes were subcloned into pVL1393 (Stratagene)for expression in Sf9 cells.

Sf9 cells were cotransfected using the BaculoGold Transfection kit(Pharmingen, San Diego, Calif.). Transfections were done according tothe manufacturer's instructions with the following modification: 8·10⁶Sf9 cells were transfected in a 100 mm dish, with 4 μg of BaculoGoldviral DNA and 6 ug of test plasmid DNA. Cells were harvested after 6days, except where otherwise specified, and assayed for VLP productionby Western Blot or ELISA assay (below).

EXAMPLE 8

Preparation of Sf9 extracts and ELISA assays.

Cells were harvested six days after transfection. Plates were scraped toresuspend cells, and the cells were collected by low speedcentrifugation. Cells were resuspended in 300 μl of breaking buffer (1MNaCl, 0.2M Tris pH 7.6) and homogenized for 30 seconds on ice using aPolytron PT 1200 B with a PT-DA 1205/2-A probe (Brinkman) in a Falcon2059 tube. Samples were spun at 2500 rpm in a GPR centrifuge (BeckmanInstruments, Inc. Palo Alto, Calif.) for 3 minutes to pellet debris.Tubes were washed with an additional 150 μl of breaking buffer,supernatents collected in a 1.5 ml microfuge tube, and respun for 5minutes in an Eppendorf microfuge (Brinkman). ELISA assays were begunthe same day.

5 μl of extract was diluted into 50 μl of 1% BSA in phosphate-bufferedsaline solution (PBS), aliquoted onto a 96 well Immulon 2 microtiterplate (Dynatech Laboratories, Inc.), and incubated overnight at 4° C.Extracts were removed and the plate blocked with 5% powdered milk/PBS.All subsequent wash steps were performed with 1% BSA/PBS. The plate wasincubated at room temperature with primary antibody for 1 hour. Theprimary antibodies, monoclonal antibodies CRPV.5A and H11.F1, wereobtained as ascites stock from Dr. Neil Christensen. They areVLP-dependent and type specific antibodies which recognize CRPV andHPV11 VLPs respectively (Neil Christiansen, personal communication).They were diluted 10⁵ -fold in 1% BSA /PBS before use. After washing in1% BSA/PBS, plates were incubated for 1 hour with secondary antibody,peroxidase labeled Goat anti-Mouse IgG (g) (Kirkegaard & PerryLaboratories, Inc.) and used at 10³ dilution in 1% BSA in PBS. After afinal washing, a horseradish peroxidase assay was performed andabsorbance read at 405 nm.

EXAMPLE 9

Transfer of the HPV11 Neutralizing Epitope to HPV6

Based upon the studies in Example 4, we mutated the HPV6 L1 gene atamino acid residues 131, 245, and 277 to match the HPV11 L1 sequence. Inaddition, we inserted a tyrosine after residue 131, extending the lengthof the mutated HPV6 L1 gene by one residue to 501 amino acids. Wedesignate this clone as HPV6:4. We predicted that these four changes,all of which match the HPV11 L1 sequence, would facilitate binding byHPV11 specific neutralizing antibodies H11.B2, H11.F1, and H11.G5. Thisis in fact true, as shown in the table below.

Relative Affinity Values to HPV6 and a Derivative

    ______________________________________                                        Antibody        HPV6    HPV6:4                                                ______________________________________                                        H11.A3          0.15    0.23                                                  H11.B2          0.18    0.82                                                  H11.F1          0.20    0.89                                                  H11.G5          0.14    0.84                                                  H11.H3          0.11    0.17                                                  ______________________________________                                    

This validates that the four amino acid residues 131,132, 245, and 277define the specificity of the binding site to neutralizing antibodiesH11.B2, H11.F1, and H11.G5.

Antibody H11.H3 can be distinguished from the other three neutralizingantibodies by sensitivity to binding at position 346, and lack ofsensitivity to binding at position 131. This indicates that the bindingof this antibody has shifted towards the C-terminus, but still overlapsthe binding site of the other three neutralizing monoclonal antibodies.

We further derivatized the HPV6 derivative clone defined above by addingan additional change at position 345, to match the sequence of HPV11 atits position 346. We designate this clone as HPV6:5. The prediction isthat it will bind all four neutralizing antibodies, including H11.H3.The data is shown in the table below.

Relative Affinity Values to HPV6 and a Derivative

    ______________________________________                                        Antibody        HPV6    HPV6:5                                                ______________________________________                                        H11.A3          0.15    0.23                                                  H11.B2          0.18    0.82                                                  H11.F1          0.20    0.89                                                  H11.G5          0.14    0.84                                                  H11.H3          0.11    0.17                                                  ______________________________________                                    

As expected, this clone produced VLPs which could bind neutralizingantibodies H11.B2, H11.F1, and H11.G5, and validates this observation.Unexpectedly, it did not bind antibody H11.H3 which indicates that anadditional change to that at position 345 is also required for bindingH11.H3.

EXAMPLE 10

Binding by neutralizing monoclonal antibody H11.H3.

To further study the binding of antibody H11.H3, a change is added atresidue 438 of the HPV6 L1 gene, to match the residue of HPV11 L1 at439. The change is added both to clone HPV6:4b (with changes at 132,245, 277 and 345) as well as HPV6:5. This will generate clong HPV5b(132, 245, 277, 345 and 435) as well as HPV6:6. Clone HPV6:5b bindsantibody H11.H3, and clone HPV6:6 binds antibodies H11.B2, H11.F1,H11.G5 and H11.H3. These clones exted the sensitivity obtainable in theassays outlined below in claims 2 and 3, and above in Examples 7 and 8.

EXAMPLE 11

To further study the binding of the neutralizing monoclonal antibodies,we back-mutated clone HPV6:4 at the four individual positions, anddemonstrated that back-mutation only at residues 131 and 132 resulted inloss of binding. Generation of an additional HPV6 L1 clone with onlythese two changes, HPV6:2 demonstrated that these two changes alone aresufficient for binding the HPV11 neutralizing monoclonal antibodies.Thus, these studies collectively define the minimal epitope for theneutralizing antibodies.

What is claimed:
 1. Synthetic virus-like particles generated from any ofthe following constructs:a) HPV6:4, wherein HPV6 L1 gene has beenmutated so that it encodes a protein wherein amino acid residues 131,245, and 277 are the same as amino acid residues 131, 246, and 278 inHPV 11 L1 protein, and after amino acid residue 131 of HPV6 L1, atyrosine residue has been inserted; b) HPV6:5, which contain the samemutations as HPV6:4 and additionally has been further mutated so that itencodes a protein which, in addition to the mutations in a), amino acidresidue 345 is the same as amino acid residue 346 in HPV11 L1 protein;c) HPV6:2, wherein HPV6 L1 gene has been mutated so that it encodes aprotein wherein amino acid 131 is the same as in HPV11 L1 protein, andadditionally a tyrosine residue has been inserted as amino acid 132; d)HPV6:4Δ132 which contains the same mutations as HPV6:4, except thatthere is no insertion of a tyrosine residue after amino acid 131; and e)HPV6:4,S131G which contains the same mutations as HPV6:4, except thatamino acid residue 131 is glycine.